High frequency application of botulinum toxin therapy

ABSTRACT

The present invention relates to methods for treating diseases and disorders by administering a composition containing the neurotoxic component of a  Clostridium botulinum  toxin complex, wherein the composition is devoid of any other protein of the  Clostridium botulinum  toxin complex and wherein the composition is administered at short intervals and/or in high doses.

FIELD OF INVENTION

The present invention relates to methods for treating diseases anddisorders by administering a composition containing the neurotoxiccomponent of a Clostridium botulinum toxin complex, wherein thecomposition is devoid of any other protein of the Clostridium botulinumtoxin complex and wherein the composition is administered at shortintervals and/or in high doses.

More particular, the present invention relates to a method of treating adisease or condition caused by or associated with hyperactivecholinergic innervation of muscles or exocrine glands in a patient, themethod comprising administering a composition comprising an effectiveamount of a neurotoxic component of a Clostridium botulinum toxincomplex, the composition being devoid of any other protein component ofthe Clostridium botulinum toxin complex, wherein (a) the patient is ahuman, (b) the composition is administered by injection, and (c) thecomposition is administered at an interval of less than three months,the interval comprising a first treatment and a second treatment,wherein the amount administered in the second treatment can be lower,higher or identical to the amount administered in the first treatment.The present invention also relates to a method of treating a disease orcondition caused by or associated with a pathological activity of amuscle in a patient, the method comprising administering a compositioncomprising an effective amount of a neurotoxic component of aClostridium botulinum toxin complex, the composition being devoid of anyother protein component of the Clostridium botulinum toxin complex,wherein (a) the patient is a human with a severe movement disorder orsevere spasticity; (b) the composition is administered by injection; and(c) the effective amount administered exceeds 500 U of neurotoxiccomponent in adults or exceeds 15 U/kg body weight in children. Finally,the present invention also relates to a method of reducing facial linesor wrinkles of the skin or of removing facial asymmetries the methodcomprising administering to an individual a composition comprising aneffective amount of a neurotoxic component of a Clostridium botulinumtoxin complex, the composition being devoid of any other proteincomponent of the Clostridium botulinum toxin complex, wherein (a) theindividual is a human; (b) the composition is administered bysubcutaneous or intramuscular injection into, or in vicinity of, one ormore facial muscles or muscles involved in the formation of the wrinkleof the skin or the asymmetry; and (c) the composition is administered atan interval of less than three months, the interval comprising a firsttreatment and a second treatment, wherein the amount administered in thesecond treatment can be lower, higher or identical to the amountadministered in the first treatment.

BACKGROUND OF INVENTION

Botulinum toxin is produced by the bacterium Clostridium. There areseven antigenically distinct serotypes of botulinum toxin, namelybotulinum toxin A, B, C, D, E, F and G. Botulinum toxins, when releasedfrom lysed Clostridium cultures are generally associated with otherbacterial proteins, which together form of a toxin complex. Theneurotoxic subunit of this complex is referred herein as the “neurotoxiccomponent” of the botulinum toxin complex. The terms “botulinum toxin”or “botulinum toxins”, refers to the neurotoxic component devoid of anyother proteins clostridial proteins, but also to the “botulinum toxincomplex”: it is used herein in cases when no discrimination between thetwo states of the neurotoxic component is necessary or desired. Thiscomplex usually contains additional so-called “non-toxic” proteins,which we will refer to as “complexing proteins” or “bacterial proteins”.The complex of neurotoxic component and bacterial proteins is referredto as “Clostridium botulinum toxin complex” or “botulinum toxincomplex”. The molecular weight of this complex may vary from about300,000 to about 900,000 Da. The complexing proteins are, for example,various hemagglutinins. The proteins of this toxin complex are not toxicthemselves but provide stability to the neurotoxic component and areresponsible for oral toxicity in botulinum intoxications. Unlike thetoxin complex, the neurotoxic component in its isolated and pure form,i.e. devoid of any complexing Clostridium proteins, is acid labile anddoes not resist the aggressive environment in the gastrointestinaltract.

The neurotoxic component of the botulinum toxin complex is initiallyformed as a single polypeptide chain, having in the case of serotype Amolecular weight of approximately 150 kDa. In other serotypes theneurotoxic component has been observed to vary between about 145 andabout 170 kDa, depending on the bacterial source. In the case ofserotype A, for example, proteolytic processing of the polypeptideresults in an activated polypeptide in the form of a dichainpolypeptide, consisting of a heavy chain and a light chain, which arelinked by a disulfide bond. In humans, the heavy chain mediates bindingto pre-synaptic cholinergic nerve terminals and internalization of thetoxin into the cell. The light chain is believed to be responsible forthe toxic effects, acting as zinc-endopeptidase and cleaving specificproteins responsible for membrane fusion (SNARE complex) (see e.g.Montecucco C., Shiavo G., Rosetto O: The mechanism of action of tetanusand botulinum neurotoxins. Arch Toxicol. 1996; 18 (Suppl.): 342-354)).By disrupting the process of membrane fusion within the cells, Botulinumtoxins prevent the release of acetylcholine into the synaptic cleft. Theoverall effect of Botulinum toxin at the neuro-muscular junction is tointerrupt neuro-muscular transmission, and, in effect, denervatemuscles. Botulinum toxin also has activity at other peripheralcholinergic synapses causing a reduction of salivation or sweating.

Each serotype of Botulinum toxin binds to the serotype specific receptorsites on the pre-synaptic nerve terminal. The specificity of Botulinumtoxin for cholinergic neurons is based on the high affinity of the heavychain for the receptor sites on these nerve terminals (Ref.: KatsekasS., Gremminloh G., Pich E. M.: Nerve terminal proteins; to fuse tolearn. Transneuro Science 1994; 17: 368-379).

Despite its toxic effects, botulinum toxin complex has been used as atherapeutic agent in a large number of diseases. Botulinum toxinserotype A was approved for human use in the United States in 1989 forthe treatment of strabism, blepharospasm, and other disorders It iscommercially available as Botulinum toxin A protein complex, forexample, under the tradename BOTOX (Allergan Inc) or under the tradenameDYSPORT (Ipsen Ltd). For therapeutic application the complex is injecteddirectly into the muscle to be treated. At physiological pH, the toxinis released from the protein complex and the desired pharmacologicaleffect takes place. The effect of Botulinum toxin is only temporary,which is the reason why repeated administration of Botulinum toxin maybe required to maintain a therapeutic affect. In a number of casesresistance to Botulinum toxin has been observed after repeatedadministration of the Botulinum toxin protein complex. Patientsdeveloped relevant levels of neutralizing antibodies directed againstthe neurotoxic component and blocking its activity (Göschel H, WohlfarthK, Frevert J, Dengler R, Bigalke H. (1997) Exp Neurol. 1997 September;147 (1):96-102. As a result, therapy with botulinum toxin complex is nolonger effective in those patients (using e.g. Botox or Dysport).Subsequent applications of Botulinum toxin medicaments such as Botox orDysport, is ineffective. The antibody titer may decrease if thetreatment with Botulinum toxin protein complexes is suspended. However,the duration for which the treatment has to be suspended may be lengthy(see, for example, Dressler D, Bigalke H (2002) Botulinum toxin antibodytitres after cessation of botulinum toxin therapy. Mov Disord17:170-173).

Initially, resistance to the Botulinum toxin complex was considereduncommon. Subsequent reports suggested a frequency of approximately 5%of antibody-induced therapy failure of botulinum toxin therapy inpatients treated for cervical dystonia (Ref.: Kessler K R, Skutta M,Benecke R., Long-term treatment of cervical dystonia with botulinumtoxin A: efficacy, safety, and antibody frequency. German Dystonia StudyGroup. J. Neurol. 1999 April; 246 (4):265-74. This observation was basedon retrospective assessments of patients at a single location. Recently,however, a much more frequent incidence has been reported, suggestingthat approximately 20% of the subjects treated are affected (Ref.: TheMuscular Nerve May 2004, p. 630). Generally, it is believed that therisk for antibody induced therapy failure is strongly correlated withthe administered single dose of botulinum toxin.

It is believed that proteins contained in the complex may reinforce theimmune response. Another risk factor for antibody-induced completefailure of botulinum toxin therapy is the interinjection interval, i.e.the interval between subsequent injection series. It is therefore commonpractise to administer Botulinum toxin only once every three months toreduce the risk of antibody formation. Patients in which the effect ofthe Botulinum administration ceases to exist earlier may be treated byoral medications. Effectiveness of those oral medications, however, islimited.

Another disadvantageous effect of Botulinum toxin protein complexes isits regional or systemic spread following injections into the targetmuscles. Single-fibre electromyography (SF-EMG) has shown increasedjitter in muscles distant from the injection site. For example, Alnty etal., 1988 (Alny R. K., Aminoff M. J., Gelb D. J., Löwenstein D. H.:Myomuscular effects distant from the site of botulinum neurotoxininjection. Neurology 1988; 38: 1780-1783) show that patients treated byinjecting into the neck muscles have increased jitter and fiber densityin muscles distant from the injection site. These abnormalities returnto normal after approximately three to six months. Other evidence ofsystemic spread of Botulinum toxin following local injection is theoccurrence of changes in cardiovascular reflexes and blood pressure.(Ref.: Alny R. K., Aminoff M. J., Gelb D. J., Löwenstein D. H.:Myomuscular effects distant from the site of botulinum neurotoxininjection Neurology 1988; 38: 1780-1783).

In some patients, the administration of botulinum toxin protein complexat high doses may affect muscles not intended for treatment. Forexample, when treating blepharospasm with a botulinum toxin proteincomplex, spreading may affect the eye lid opening muscle causing ptosis.

Therefore, not only the risk of antibody formation but also the risk ofsystemic spread makes it necessary to administer botulinum toxin atcomparatively low and thus potentially less effective doses.Consequently, physicians are strongly advised to administer BOTOX orDYSPORT not more often than once every three months. This applies inparticular to patients that require high doses of Botulinum toxin.

In view of the above, it is an objective of the invention to provide atreatment for patients affected by the disorders mentioned herein, thatallows administration of a therapeutic botulinum toxin preparation atreduced intervals and/or by using comparatively high doses. Sincetherapeutic botulinum toxin preparations are to be applied in specifictarget tissues (e.g. specific muscles or glands), it is an importantrequirement that its spread into neighboring tissue is limited. A finalbut not less important requirement is a reduced antigenicity of thetherapeutic botulinum toxin preparation. It is also an objective of theinvention to provide methods for treating a disease associated with aspastic or dystonic muscle with a suitable medicament at flexible and/orfrequent intervals. Another objective of the invention is to provide acosmetic treatment using the drug at frequent intervals with a reducedrisk of antibody formation and/or reduced systemic spread.

Accordingly, the present invention relates to a method of treating adisease or condition caused by or associated with hyperactivecholinergic innervation of muscles or exocrine glands in a patient, themethod comprising administering a composition comprising an effectiveamount of a neurotoxic component of a Clostridium botulinum toxincomplex, the composition being devoid of any other protein component ofthe Clostridium botulinum toxin complex, wherein (a) the patient is ahuman, (b) the composition is administered by injection, and (c) thecomposition is administered at an interval of less than three months,the interval comprising a first treatment and a second treatment,wherein the amount administered in the second treatment can be lower,higher or identical to the amount administered in the first treatment.

It is noteworthy that the concept of the present invention, whichinvolves the administration of the neurotoxic component of the botulinumtoxin complex, generally allows the treatment of any condition which isassociated with hyperactive cholinergic innervation of a muscle or anexocrine gland, where the neurotoxic component blocks acetylcholinesecretion into the synaptic cleft. Therefore, treatment offered by thepresent invention may be directed at any of the following indications,most of which are described in detail in Dressler D (2000) (BotulinumToxin Therapy. Thieme Verlag, Stuttgart, New York):

dystonia

-   -   cranial dystonia        -   blepharospasm        -   oromandibular dystonia            -   jaw opening type            -   jaw closing type        -   bruxism        -   Meige syndrome        -   lingual dystonia        -   apraxia of eyelid opening    -   cervical dystonia        -   antecollis        -   retrocollis        -   laterocollis        -   torticollis    -   pharyngeal dystonia    -   laryngeal dystonia        -   spasmodic dysphonia/adductor type        -   spasmodic dysphonia/abductor type        -   spasmodic dyspnea    -   limb dystonia        -   arm dystonia            -   task specific dystonia                -   writer's cramp                -   musician's cramps                -   golfer's cramp        -   leg dystonia            -   thigh adduction, thigh abduction            -   knee flexion, knee extension            -   ankle flexion, ankle extension            -   equinovarus deformity        -   foot dystonia            -   striatal toe            -   toe flexion            -   toe extension        -   axial dystonia            -   pisa syndrome            -   belly dancer dystonia        -   segmental dystonia        -   hemidystonia        -   generalised dystonia    -   dystonia in lubag    -   dystonia in corticobasal degeneration    -   dystonia in lubag    -   tardive dystonia    -   dystonia in spinocerebellar ataxia    -   dystonia in Parkinson's disease    -   dystonia in Huntington's disease    -   dystonia in Hallervorden Spatz disease    -   dopa-induced dyskinesias/dopa-induced dystonia    -   tardive dyskinesias/tardive dystonia    -   paroxysmal dyskinesias/dystonias        -   kinesiogenic        -   non-kinesiogenic        -   action-induced            palatal myoclonus            myoclonus            myokymia            rigidity            benign muscle cramps            hereditary chin trembling            paradoxic jaw muscle activity            hemimasticatory spasms            hypertrophic branchial myopathy            maseteric hypertrophy            tibialis anterior hypertrophy            nystagmus            oscillopsia            supranuclear gaze palsy            epilepsia partialis continua            planning of spasmodic torticollis operation            abductor vocal cord paralysis            recalcitant mutational dysphonia            upper oesophageal sphincter dysfunction            vocal fold granuloma            stuttering            Gilles de la Tourette syndrome            middle ear myoclonus            protective larynx closure            postlaryngectomy speech failure            protective ptosis            entropion            sphincter Odii dysfunction            pseudoachalasia            nonachalsia oesophageal motor disorders            vaginismus            postoperative immobilisation            tremor            bladder dysfunction    -   detrusor sphincter dyssynergia    -   bladder sphincter spasm        hemifacial spasm        reinnervation dyskinesias        cosmetic use    -   crow's feet    -   frowning    -   facial asymmetries    -   mentalis dimples        stiff person syndrome        tetanus        prostate hyperplasia        adipositas treatment        infantile cerebral palsy        strabismus    -   mixed    -   paralytic    -   concomitant    -   after retinal detachment surgery    -   after cataract surgery    -   in aphakia    -   myositic strabismus    -   myopathic strabismus    -   dissociated vertical deviation    -   as an adjunct to strabismus surgery    -   esotropia    -   exotropia        achalasia        anal fissures        exocrine gland hyperactivity    -   Frey syndrome    -   Crocodile Tears syndrome    -   hyperhidrosis        -   axillar        -   palmar        -   plantar            rhinorrhea            relative hypersalivation    -   in stroke    -   in parkinsosn's    -   in amyotrophic lateral sclerosis        spastic conditions    -   in encephalitis and myelitis        -   autoimmune processes            -   multiple sclerosis            -   transverse myelitis            -   Devic syndrome        -   viral infections        -   bacterial infections        -   parasitic infections        -   fungal infections    -   in hereditary spastic paraparesis    -   postapoplectic syndrome        -   hemispheric infarction        -   brainstem infarction        -   myelon infarction    -   in central nervous system trauma        -   hemispheric lesions        -   brainstem lesions        -   myelon lesion    -   in central nervous system hemorrhage        -   intracerebral hemorrhage        -   subarachnoidal hemorrhage        -   subdural hemorrhage        -   intraspinal hemorrhage    -   in neoplasias        -   hemispheric tumors        -   brainstem tumors        -   myelon tumors

Botulinum toxin is obtainable, for example, by cultivation ofClostridium bacteria. The preferred Clostridium species of the presentinvention is Clostridium botulinum. However, it is important to notethat the neurotoxic component may be derived from any other bacterialspecies. Provided it is a functional homolog of the neurotoxic componentderived from Clostridium botulinum. The composition used in the methodsof the present invention will always contain the neurotoxic componentdevoid of any other Clostridium botulinum proteins. However, whenproducing the neurotoxic component, the toxin may be isolated from thebacteria as a complex containing the neurotoxic component, i.e. theprotein responsible for the toxic effect in humans and other bacterialproteins. Subsequently the neurotoxic component may be purified from thetoxin complex. As used herein, the terms “toxin complex” or “botulinumtoxin complex” or “botulinum neurotoxin complex” are interchangeable andrefer to a high molecular weight complex comprising the neurotoxiccomponent of approximately 150 kDa and, in addition, non-toxic proteinsof Clostridium botulinum, including hemagglutinin and non-hemagglutininproteins (Sakaguchi 1983; Sugiyama 1980).

The present invention envisages treating patients characterized byhaving a disease associated with hyperactive cholinergic innervation ofmuscles or exocrine glands. The term “patient” as used herein refers topatients who have never been exposed to botulinum toxin but also topatients who have been exposed to botulinum toxin. The latter patientmay have developed antibodies directed against the botulinum toxincomplex or its components. Such antibodies may be neutralizingantibodies. Preferably, the patients do not have an antibody titer above7 mU, in particular a titer of neutralizing antibodies above 7 mU. Theterm “antibody titer not above . . .” means less than 7 mU, e.g. 1 mU to6 mU or 0.01 mU to 1 mU.

The term “hyperactive cholinergic innervation”, as used herein, relatesto a synapse, which is characterized by an unusually high amount ofacetylcholine release into the synaptic cleft. “Unusually high” relatesto an increase of up to 25%, up to 50% or more with respect to areference activity which may be obtained, for example, by comparing therelease with the release at a synapse of the same type but which is notin a hyperactive state, wherein muscle dystonia may be indicative of thehyperactive state. “Up to 25%” means, for example, about 1% to about25%. Methods for performing the required measurements are known in theart.

The term “about” as used in the context of the present invention means“approximately” or “nearly”. In the context of numerical values, withoutcommitting to a strict numerical definition, the term may be construedto estimate a value that is +/−10% of the value or range indicated.

The terms “neurotoxic component” or “neurotoxin component” as usedthroughout the specification, relates to the subunit of the botulinumtoxin complex which has a neurotoxic activity and which has a molecularweight of approximately 150 kDa in serotype A. The term “neurotoxiccomponent” also includes the functional homologs found in the otherserotypes of Clostridium botulinum. In a preferred embodiment of thepresent invention the neurotoxic component is devoid of any other C.botulinum protein, preferably also of RNA potentially associated withthe neurotoxic component. The neurotoxic component may be the singlechain precursor protein of approximately 150 kDa or the proteolyticallyprocessed neurotoxic component, comprising the light chain (L_(c)) ofapproximately 50 kDa and the heavy chain (H_(c)) of approximately 100kDa, which may be linked by one or more disulfide bonds (for a reviewsee e.g. Simpson L L, Ann Rev Pharmacol Toxicol. 2004; 44:167-93). Thoseof skill in the art will appreciate that full biological activity isattained only after proteolytic activation, even though it isconceivable that the unprocessed precursor can exert some biologicalfunctions or be partially active. “Biological activity” refers to (a)receptor binding, (b) internalization, (c) translocation across theendosomal membrane into the cytosol, and/or (d) endoproteolytic cleavageof proteins involved in synaptic vesicle membrane fusion. In vitroassays for assessing biological activity include the mouse LD₅₀ assayand the mouse hemidiaphragm assay as described by Pearce L B, Borodic GE, First E R, MacCallum R D (1994) (Measurement of botulinum toxinactivity: evaluation of the lethality assay. Toxicol Appl Pharmacol 128:69-77) and Dressler D, Lange M, Bigalke H (2005) (The mouse diaphragmassay for detection of antibodies against botulinum toxin type B. MovDisord 20:1617-1619).

The biological activity is commonly expressed in Mouse Units (MU). Asused herein, 1 MU is the amount of neurotoxic component, which kills 50%of a specified mouse population after intraperitoneal injection, i.e.the mouse i.e. LD₅₀ (Schantz & Kauter, 1978).

The terms “MU” and “Unit” or “U” are interchangeable. Alternatively, thebiological activity may be expressed in Lethal Dose Units (LDU)/ng ofprotein (i.e. neurotoxic component). The term “MU” is used hereininterchangeably with the terms “U” or “LDU”.

The term “effective amount” means an amount of neurotoxic component,which, after administration, results in a partial or complete removal ofdisease symptoms. Effective amounts are generally in the range of 1 to2000 MU but also doses of up to 5000 MU may be used. When high doses ofneurotoxic component are to be administered to a patient, it may bebeneficial to split the treatment into more than one treatment session.The term “more than one treatment session” means e.g. 2, 3, 4, 5, 6, 7,8, 9 treatment sessions. Preferably, the neurotoxic component used inthe methods of the present invention is purified from a culture of C.botulinum. Methods for cultivating C. botulinum and purifying the toxincomplex therefrom have been described in the art (Reviewed in Schantz &Kauter 1978. Microbiological methods. Standardized assay for Clostridiumbotulinum neurotoxins J Assoc Off Anal Chem 1978; 61 (1):96-99.) Theneurotoxic component may be purified from C. botulinum essentially asdescribed in the method of DasGupta & Sathyamoorthy (DasGupta B R,Sathyamoorthy V. Purification and amino acid composition of type Abotulinum neurotoxin. Toxicon. 1984; 22 (3):415-24. To this end,Clostridium botulinum type A is cultivated for example in a 20 lfermentor in a medium consisting of 2% proteose peptone, 1% yeastextract, 1% glucose and 0.05% sodium thioglycolate. After growth for 72hours, the toxin is precipitated by adding 3 N sulfuric acid (finalpH=3.5). The precipitated and centrifuged biomass is extracted with 0.2M sodium phosphate buffer at pH 6.0. After removal of the nucleic acidsby precipitation with protamine sulfate the toxin is precipitated byadding ammonium sulfate. The precipitate which has been solubilized anddialyzed against 50 mM sodium phosphate at pH 6.0 is bound to aDEAE-Sephadex® column at the same pH and eluted with 150 mM NaCl. Thisis followed by a chromatography on a QAE-Sephadex® column which has beenequilibrated with a 50 mM Tris/HCl buffer pH 7.9. The toxin is elutedvia a NaCl gradient. In the last step, the toxin is chromatographed onSP-Sephadex® at pH 7.0. In this case, the bound toxin is eluted from thecolumn using a NaCl gradient (0-300 mM). The purified toxin is analyzedby SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and generallyexhibits a purity of 95+/−5%.

Botulinum neurotoxins, in particular the toxin complex described above,have previously been classified into seven serologically distinct typesA, B, C, D, E, F and G. In recent years, distinct populations of theA-(A1 and A2) and C-serotypes (C1 and C2) have been identified. Herein,these populations are designated as “subtypes”.

The neurotoxic component of serotype A is commercially available underthe trade name XEOMIN from Merz Pharmaceuticals in a composition that isdevoid of any other proteins of the Clostridium botulinum toxin complex.

Alternatively, the neurotoxic component used in the methods of thepresent invention may be generated by recombinant gene expression. Tothis end, an open reading frame encoding the neurotoxic component or amutant thereof may be cloned into a vector adapted for gene expressionin a host cell of interest. Methods for recombinant gene expression andprotein purification are known to the person skilled in the art.

The recombinant nucleic acid molecule encoding the neurotoxic componentmay be derived from a known nucleic acid sequence or may be recombinedfrom two or more known sequences by recombinant techniques or chemicalsynthesis. An example of a chimeric neurotoxic component is a moleculegenerated by fusing e.g. the light chain of a first serotype to theheavy chain of a second serotype of the neurotoxic component. An exampleof chemical synthesis is the chemical synthesis of the entire neurotoxiccomponent.

Also included are genetically modified neurotoxic components containing1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 20 amino acid mutations. Amutation may be a substitution, an insertion or a deletion. Preferably,the mutation does not compromise any of the biological activitiesindicated above. However, it is also envisaged to use mutations tomodulate the biological activity of the neurotoxic component.

Also included are the neurotoxic components of Botulinum toxinscontaining chemically modified amino acids, for example one or moreamino acids which are glycosylated, acetylated or otherwise modified,which may be beneficial to the uptake or stability of the toxin.Particularly preferred is the lipidation of the neurotoxic component.Modifying residues may be added to the neurotoxic component e.g. bymeans of an enzymatic in vitro reaction or by using appropriate chemicalreaction conditions. Alternatively, modifying enzymatic functions may beprovided in trans by expressing the enzyme within the host cell.

Using the method described above allows significantly increasing thefrequency of treatment without inducing neutralizing antibodies directedagainst the neurotoxic component. In this regard, it should be notedthat prior to the present invention, known treatment regimens strictlyavoided administration of botulinum toxin at intervals of less thanthree months, since a more frequent administration of botulinum toxinwas thought to increase the likelihood of inducing an immune response inthe patient treated. The examples disclosed herein support the notionthat the use of the neurotoxic component instead of the botulinum toxincomplex can avoid such problems.

In a preferred embodiment of the present invention, the second treatmentis performed in order to improve the treatment effect of the firsttreatment. This will allow administration of appropriate botulinum toxindoses more efficiently. For example, in a first treatment session asuboptimal dose of the neurotoxic component may be administered. Shouldthe patient's disease symptoms not sufficiently respond, more neurotoxiccomponent may be administered in a second or in subsequent treatmentsession(s). Therefore, in view of the reduced risk associated with themethods of the present invention, a number of treatment sessions may beused in order to approach the optimal dose necessary to effectivelytreat a patient.

In accordance with the present invention, a first and a second and asubsequent treatment session may be scheduled at least one day after apreceding treatment session. The term “at least one day after” meanse.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 weeks. However, it is also envisaged by the teaching of thepresent invention that the second treatment is scheduled only few hoursafter the first treatment, e.g. 2, 3, 4, 5, 6, 7 or 8 hours later.

In a preferred embodiment, the second (subsequent) treatment is carriedout at a point in time when the efficacy of the first (previous)treatment begins to decline. With such a treatment regimen, a stablequality of life for the patients can be achieved.

The determination of the parameter “stable quality of life” for thepatients is exemplarily described hereinunder by reference to onecondition to be treated according to the present invention, namelyblepharospasm, on the basis of the so-called Blepharospasm DisabilityIndex (BSDI). The Blepharospasm Disability Index [BSDI] is a self-ratingscale for assessment of impairment of specific activities of dailyliving caused by BEB Goertelmeyer R, Brinkmann S, Comes G, Delcker A,The Blepharospasm Disability Index (BSDI) for the Assessment ofFunctional Health in Focal Dystonia, Clin. Neurophysiol. 2002; 113 (1):S77-S78.

The scale is to be answered by the patient at each visit. It includes 6items to be assessed with a 5-point listing (i.e., 0-4 points per item)ranging from “no impairment, “slight/moderate/severe impairment” and “nolonger possible due to my illness”. The 6 items are “Driving a vehicle”,“Reading”, “Watching TV”, “Shopping”, “Walking” and “Doing everydayactivities”. Unlike other functional scales, which ignore scaling incase of non-applicable items, the BSDI allows for answering as ‘notapplicable’ for five items except “Doing everyday activities”

The BSDI mean score for non-missing items is calculated by adding allapplicable and answered items, and dividing by the number of itemsanswered.

However, such parameters are available for many other diseases andconditions to be treated within the present invention, e.g.craniocervical dystonia questionnaire (CCDQ 24) for cervical dystonia(Mueller J, Wissel J, Kemmler G, Bodner T, Poewe W, Quality of life inpatients with craniocervical dystonia: development of the CCDQ-24, Mov.Disord. 2000; 15 (Suppl 3): 761, and HRQL, by the Swedish Short Form 36Health Survey Questionnaire (SF-36) for spasticity (Welmer A K, vonArbin M, Widen Holmqvist L, Sommerfeld D K, Spasticity and itsassociation with functioning and health-related quality of life 18months after stroke, Cerebrovasc. Dis. 2006; 21 (4): 247-253).

At each of the re-injection treatment or last treatment of the patient,the difference Δ_(BSDI) between the actual BSDI observation and the BSDIvalue at baseline will be calculated:Δ_(BSDI)=BSDI_(actual)−BSDI_(baseline)

The BSDI_(baseline) is determined during the first visit of the patientto be treated and before the first injection of the medicament. TheBSDI_(actual) is determined after the respective (re)-injection of themedicament and 3 weeks thereafter, respectively.

On the basis of the Δ_(BSDI) value recorded at the first treatment, eachpatient will be allocated to one of three strata in the following way:Stratum 1 (moderate improvement): −1.00 ≦ Δ_(BSDI) ≦ −0.65 Stratum 2(marked improvement): −1.35 ≦ Δ_(BSDI) < −1.00 Stratum 3 (abolishment ofsigns Δ_(BSDI) < −1.35 and symptoms):

A patient is classified as a responder, i.e., the patient demonstrates astable level of quality of life, if none of the Δ_(BSDI) valuescalculated exceeds a threshold Δ_(c). The value of the threshold Δ_(c)depends on the stratum the patient belongs to. The values of thethresholds are: Stratum 1: Δ_(c) = −0.40 Stratum 2: Δ_(c) = −0.75Stratum 3: Δ_(c) = −1.10

Responders show a reduction of their baseline BSDI value, and thereforean improvement of their quality of life status. The minimal magnitude ofthe improvement is given by the threshold Δ_(c). The values of Δ_(c)decrease with the number of the stratum because patients in Stratum 2show a stronger response to the initial injection than patients inStratum 1 (resulting in lower BSDI differences), and patients in Stratum3 react even stronger than patients in Stratum 2.

Finally, the observed differences between the BSDI level at the day of are-injection treatment and the baseline BSDI level will be analyzed toinvestigate if there is any improvement over time of the quality of lifelevel at the time of re-injection (expected time of waning of treatmenteffect).

As used throughout the specification of the present invention, the term“total amount injected per treatment” refers to the total dosage andmeans the sum of neurotoxin applied to a patient during a singletreatment. A single treatment may involve one or more injections Forexample the treatment of M. sternocleidomastoideus, M. splenius capitis,M. semispinalis capitis and M. trapezius may involve 1, 2, 3, 4 or 5injections, whereas the treatment of M. levator scapulae or Mm scalenimay involve only 1 to 3 injections. As pointed out herein before, theamount used for treatment is dependent on a number of parameters, whichare known in the art. Such parameters include for example the sero-typeof the neurotoxic component, the target tissue to be injected and anumber of patient specific factors. It is envisaged by the teaching ofthe present invention that a single treatment may be split into two ormore treatment sessions during which the above mentioned total amount ofneurotoxic component is administered. This will be particularly the caseif large amounts of neurotoxic component are to be administered.

Moreover, based on this embodiment of the present invention's method itwill now be possible to more effectively treat a patient in need of anadditional administration of the neurotoxic component. This may be thecase, e.g. when, after a first or previous treatment it is establishedthat additional muscles contribute to the disease symptoms or whenmuscles have been missed.

In another preferred embodiment of the present invention, the patient isa patient requiring high doses of neurotoxic component. In anotherpreferred embodiment of the present invention, (a) the patient is apatient with a severe movement disorder or severe spasticity and (b) theeffective amount administered exceeds 500 U of neurotoxic component inadults or exceeds 15 U/Kg body weight in children.

Based on the embodiment of the present invention, it is now possible totreat patients with far greater amounts of neurotoxic component. Inadult patients, such amounts may for example exceed 500 U of neurotoxiccomponent.

As used throughout the present invention, an amount exceeding 500 U isfor example an amount of more than 500 U and up to 550 U, up to 600 U,up to 700 U, up to 800 U, up to 900 U, up to 1000 U, up to 1100 U, up to1200 U, up to 1300 U, up to 1400 U, up to 1500 U, up to 1600 U, up to1700 U, up to 1800 U, up to 1900 U, or up to 2000 U. Preferably the doseadministered is in the range of 500 to 900 U, more preferablyapproximately 850 U. In children, “high amounts” means amounts exceeding15 U/kg and up to 16 U/kg, up to 17 U/kg, up to 18 U/kg, up to 19 U/kg,up to 20 U/kg.

In a more preferred embodiment of the present invention, the amountwhich exceeds 500 U is a total amount in adults or 15 U/kg body weightin children and the amount is administered by (a) injecting a firstfraction of this amount during a first treatment session and (b)injecting the remaining fraction during one or more subsequent treatmentsession(s), wherein the subsequent treatment session(s) is/are scheduledat least one day after the first treatment session. The total effectiveamount of neurotoxic component may be administered on the same day or ondifferent days, i.e. in different treatment sessions. Preferably, whenhigh amounts of neurotoxic component are to be administered, the totalamount to be administered may be split and administered in two or moretreatment sessions This way, large amounts, which would otherwise not becompliant when administered in a single treatment session, may beadministered to a patient without observing significant adverse effects.

In another preferred embodiment of the present invention, the patient isa human, who has been treated with Botulinum toxin but who complainsabout a decrease of the treatment effect and who requires treatmentbefore expiry of 3 months after the treatment.

Such decreases of the therapeutic effect can be monitored by treatmentcalendars in which the patient records the severity of his disorder on aday-to-day basis (such treatment calendars are, for example, distributedby Merz Pharmaceuticals).

In yet another preferred embodiment of the present invention, thehyperactive gland is an autonomic exocrine gland and the composition isinjected into or in the vicinity of that gland.

In a preferred embodiment, the autonomous exocrine gland is (a) selectedfrom the group consisting of sweat gland, tear gland, salivary gland andmucosal gland; or (b) a hyperactive gland which is associated with adisease or condition selected from the group consisting of Freysyndrome, Crocodile Tears syndrome, axillar hyperhidrosis, palmarhyperhidrosis, plantar hyperhidrosis, hyperhidrosis of the head andneck, hyperhidrosis of the body, rhinorrhea, or relative hypersalivationin patients with stroke, Parkinson's disease or Amyotrophic LateralSclerosis. It is, however, to be noted that the target tissue of therapyof the neurotoxic component covers any exocrine gland with hyperactivityAccordingly, it is envisaged that the present invention can be appliedto the treatment involving any of the glands mentioned in Sobotta,Johannes: (Atlas der Anatomie des Menschen. 22. Auflage. Band 1 und 2.Urban & Fischer, 2005), which is incorporated herein by reference.

The present invention also relates to a method of treating a disease orcondition caused by or associated with a pathological activity of amuscle in a patient, the method comprising administering a compositioncomprising an effective amount of a neurotoxic component of aClostridium botulinum toxin complex, the composition being devoid of anyother protein component of the Clostridium botulinum toxin complex,wherein (a) the patient is a human with a severe movement disorder orsevere spasticity; (b) the composition is administered by injection; and(c) the effective amount administered exceeds 500 U of neurotoxiccomponent in adults or exceeds 15 U/kg body weight in children.

In a preferred embodiment of the present invention, the amount whichexceeds 500 U is a total amount in adults or 15 U/kg body weight inchildren and wherein the amount is administered by (a) injecting a firstfraction of this amount during a first treatment session and (b)injecting the remaining fraction during one or more subsequent treatmentsession(s), wherein the subsequent treatment session is scheduled atleast one day after the first treatment session.

In another preferred embodiment of the present invention, thecomposition is administered at an interval of less than three months,the interval comprising a first treatment and a second treatment,wherein the amount administered in the second treatment can be lower,higher or identical to the amount administered in the first treatment.

In yet another preferred embodiment of the present invention, thedisease or condition is or involves dystonia of a muscle. The term“disease or condition which is or involves dystonia or dystonia of amuscle” refers to a condition involving a dystonic muscle. Preferablythe condition is selected from the group consisting of generalizeddystonia, segmental dystonia, focal dystonia, multifocal dystonia andhemidystonia. Focal dystonia is preferably selected from the groupconsisting of cranial dystonia, cervical dystonia, dystonia of thelimbs, dystonia of the trunk, and spasmodic dysphonia. Cervicaldystonia, also referred to as spasmodic torticollis, is characterized byinvoluntary, inappropriate muscle hyperactivity in muscles of the neckand the shoulder, leading to abnormal head movements and postures, jerksor tremor (Fahn S. Assessment of the Primary Dystonias In: Munsat T L,editor. Quantification of Neurologic Deficit. Boston: Butterworths;1989. p. 241-270. (ID 1760137)). Injection of the neurotoxic componentin the affected neck and shoulder muscles leads to a significant reliefof symptoms in most patients.

In a more preferred embodiment of the present invention, the dystonia is(a) selected from the group consisting of (1) cranial dystonia includingblepharospasm, oromandibular dystonia of the jaw opening or jaw closingtype, bruxism, Meige syndrome, lingual dystonia apraxia of eyelidopening, (2) cervical dystonia including antecollis, retrocollis,laterocollis, torticollis (3) pharyngeal dystonia, (4) laryngealdystonia including spasmodic dysphonia of the adductor type or of theabductor type, spasmodic dyspnea, (5) limb dystonia including armdystonia such as task specific dystonias, including writer's cramp,musician's cramps or golfer's cramp, leg dystonia involving thighadduction, thigh abduction knee flexion, knee extension, ankle flexion,ankle extension or equinovarus deformity foot dystonia involvingstriatal toe, toe flexion or toe extension, axial dystonia such as Pisasyndrome or belly dancer dystonia, segmental dystonia, hemidystonia orgeneralised dystonia, (6) dystonia in Lubag, (7) dystonia incorticobasal degeneration (8) tardive dystonia, (9) dystonia inspinocerebellar ataxia, (10) dystonia in Parkinson's disease, (11)dystonia in Huntington's disease, (12) dystonia in Hallervorden Spatzdisease, (13) dopa-induced dyskinesias/dopa-induced dystonia, (14)tardive dyskinesias/tardive dystonia, (15) paroxysmaldyskinesias/dystonias (kinesiogenic, non-kinesiogenic, action-induced);or (b) involves a clinical pattern selected from the group consisting oftorticollis, laterocollis retrocollis, anterocollis, flexed elbow,pronated forearm, flexed wrist, thumb-in-palm or clenched fist.

The following table provides a non-limiting list of clinical patternsand the muscles potentially involved, the muscles being preferred targetmuscles in accordance with the teaching of the present invention.Clinical Pattern Potential Target Muscles Torticollis splenius capitis,sternocleidomastoid, trapezius Laterocollis sternocleidomastoid,splenius capitis, scalene complex, levator scapulae Retrocollis spleniuscapitis, trapezius-pars cervicalis Anterocollis sternocleidomastoid,scalene complex, submental complex, suprahyoidal and infrahyoidalmuscles

“Spasmodic dysphonia” is a voice disorder caused by involuntarymovements of one or more muscles of the larynx. Patients affected byspasmodic dysphonia have difficulty talking. Spasmodic dysphonia causesthe voice to break or to have a tight, strained, strangled or effortfulquality. As disclosed herein, injection of the neurotoxic component intothe affected muscles of the larynx generally improves the voice.

Blepharospasm, is a progressive disease characterized by spontaneous,bilateral, intermittent or persistent involuntary contractions of theorbicular oculi muscles (Grandas F, Elston J, Quinn N, Marsden C D.Blepharospasm: A review of 264 patients. J Neurol Neurosurg Psychiatry1988; 51 (6): 767-772. (ID 1759120); Jankovic J, Orman J. Blepharospasm:Demographic and clinical survey of 250 patients. Ann Opthalmol 1984; 16(4): 371-376. (ID 1761786); Mauriello J A, Leone T, Dhillon S, PakemanB, Mostafavi R, Yepez M C. Treatment choices of 119 patients withhemifacial spasm over 11 years. Clin Neurol Neurosurg 1996; 98 (3):213-216. (ID 1777068)). Given as a local injection in the orbicularisoculi muscles, on the basis of the methods of the present invention,Botulinum toxin is a highly effective and well tolerated symptomatictreatment of blepharospasm.

In another preferred embodiment, patients with benign essentialblepharospasm who are pre-treated with botulinum toxin type A who show ashort duration of efficacy, are treated with botulinum neurotoxin freeof complexing proteins by administration of said botulinum neurotoxin inshortened injection intervals, i.e. in intervals of less than threemonths. Preferably, the botulinum neurotoxin free of complexing proteinsis highly purified botulinum neurotoxin type A. A new injection withbotulinum neurotoxin is indicated when the patient reports a decline intreatment effect. Due to said treatment regimen, said patients canachieve a stable quality of life as discussed hereinbefore, which ispreferably determined as discussed hereinbefore.

In a more preferred embodiment of the present invention, the muscle isselected from the group consisting of Ipsilateral splenius,contralateral sternocleidomastoid, ipsilateral sternocleidomastoidsplenius capitis, scalene complex, levator scapulae, postvertebralis,ipsilateral trapezius, levator scapulae, bilateral splenius capitis,upper trapezius, deep postvertebralis, bilateral sternocleidomastoid,scalene complex, submental complex, brachioradialis, bicepsbrachialis,pronator quadratus, pronator teres, flexor carpi radialis, flexor carpiulnaris, flexor pollicis longus adductor pollicis, flexor pollicisbrevis/opponens flexor digitorum superficialisflexor digitorumprofundus.

In a preferred embodiment of the present invention, the disease orcondition is or involves spasticity of a muscle.

In a more preferred embodiment of the present invention, the spasticityis or is associated with (a) post-stroke spasticity, spasticity causedby cerebral palsy; or (b) (1) a spastic condition in encephalitis andmyelitis relating to (a) autoimmune processes including respect tomultiple sclerosis, transverse myelitis, Devic syndrome, (b) viralinfections, (c) bacterial infections, (d) parasitic infections or (e)fungal infections, (2) hereditary spastic paraparesis, (3)postapoplectic syndrome resulting from hemispheric infarction, brainsteminfarction or, myelon infarction, (4) a central nervous system traumainvolving e.g. a hemispheric lesion, a brainstem lesion, a myelonlesion, (5) a central nervous system hemorrhage such as an intracerebralhemorrhage, a subarachnoidal hemorrhage, a subdural hemorrhage or anintraspinal hemorrhage, or (6) a neoplasia, e.g. a hemispheric tumor, abrainstem tumors or a myelon tumor. Other treatments may be thetreatment of urinary bladder, spastic bladder, incontinence, spasticsphincter, spasticity caused by cerebral palsy or prostatic hyperplasia.

The term “post-stroke spasticity” relates to spasticity occurring aftera stroke incident. Stroke is a leading cause of long-term disability,with spasticity occurring in 19% (2) to 38% of patients (Watkins C L,Leathley M J, Gregson J M, Moore A P, Smith T L, Sharma A K. Prevalenceof spasticity post stroke. Clin Rehabil 2002; 16 (5): 515-522. (ID1915001)). Spasticity is defined as a motor disorder characterized by avelocity-dependent increase in tonic stretch reflexes (muscle tone) withexaggerated tendon jerks, resulting from hyperexcitability of thestretch reflex, as one component of the upper motor neuron syndrome (4).In some patients spasticity can be beneficial, as in the case of hip andknee extensor spasticity, which may allow weight bearing, with theaffected limb acting like a splint (5). However, in the majority ofpatients spasticity causes difficulties with activities of daily living,such as dressing and cleaning the palm of the clenched hand (6). Inaccordance with the teaching of the present invention, common clinicalpatterns of deformity associated with spasticity in the correspondingmuscle groups are treated with the neurotoxic component.

The term “urinary bladder” relates to a disorder of the bladder often,but not necessarily, resulting from a spinal cord lesion or multiplesclerosis or trauma resulting in incontinence and deteriorated voidingof urine. Preferably, the target muscle of neurotoxin administration isthe striated sphincter urethrae muscle as described elsewhere (SchurchB. The role of botulinum toxin in neurourology. Drugs Today 2004; 40(3): 205-212. (ID 3097145); Schurch B, De Sèze M, Denys P,Chartier-Kastler E, Haab F, Everaert K, et al. Botulinum toxin type a isa safe and effective treatment for neurogenic urinary incontinenceresults of a single treatment, randomized, placebo controlled 6-monthstudy. J Urol 2005; 174 (1): 196-200. (ID 3528462)).

The term “incontinence” means urinary incontinence, which is theinability to control the flow of urine from the bladder. There arevarious kinds and degrees of incontinence, which are within the scope ofthe teaching of the present invention: overflow incontinence is acondition in which the bladder retains urine after voiding; as aconsequence, the bladder remains full most of the time, resulting ininvoluntary seepage of urine from the bladder; stress incontinence isthe involuntary discharge of urine when there is increased pressure uponthe bladder, as in coughing or straining to lift heavy objects; totalincontinence is the inability to voluntarily exercise control over thesphincters of the bladder neck and urethra, resulting in total loss ofretentive ability. In the treatment of incontinence the compositionmentioned herein may for example be injected cystoscopically into thedetrusor muscle, excluding the trigonal region (Schurch B. The role ofbotulinum toxin in neurourology. Drugs Today 2004; 40 (3): 205-212. (ID3097145); Schurch B, De Sèze M, Denys P, Chartier-Kastler E, Haab F,Everaert K, et al. Botulinum toxin type a is a safe and effectivetreatment for neurogenic urinary incontinence: results of a singletreatment, randomized, placebo controlled 6-month study. J Urol 2005;174 (1): 196-200. (ID 3528462)).

The term “prostatic hyperplasia” refers to an enlargement of theprostate in which the normal elements of the prostate gland grow in sizeand number. Their sheer bulk may compress the urethra, which coursesthrough the center of the prostate, impeding the flow of urine from thebladder through the urethra to the outside. This may lead to urineretention and the need for frequent urination. If prostatic hyperplasiais severe, complete blockage may occur. After injecting the compositiondescribed herein into the prostate, a significant reduction of symptoms,serum markers of the prostate, prostate volume, post-void residual urinevolume, and peak urinary flow rates are observed. Similar results havebeen described elsewhere (Maria G, Brisinda G, Civello I M, BentivoglioA R, Sganga G, Albanese A. Relief by botulinum toxin of voidingdysfunction due to benign prostatic hyperplasia: results of arandomized, placebo-controlled study. Urology 2003; 62 (2): 259-265. (ID2562820)).

“Cerebral palsy” describes a wide spectrum of pyramidal dysfunctionscausing paresis, extrapyramidal dysfunctions causing dystonia, rigidity,spasticity and spasms, apraxic components and coordinative dysfunctions.Cerebral palsy (Koman L A, Mooney J F, Smith B P, Goodman A, Mulvaney T.Management of spasticity in cerebral palsy with botulinum—A toxin:report of a preliminary, randomized, double-blind trial. J PediatrOrthop 1994; 14 (3): 299-303. (ID 1767458); Pidcock F S. The emergingrole of therapeutic botulinum toxin in the treatment of cerebral palsy.J Pediatr 2004; 145 (2 Suppl): S33-S35. (ID 2994781)) may occur afterbrain hemorrhage, asphyxia, premature birth and other perinatalcomplications. It is a life-long condition causing uncoordinatedmovements paresis and various forms of muscle hyperactivity. Patientsaffected by cerebral palsy, when treated in accordance with the methodsdisclosed herein, experience a functional improvement of hyperactivemuscles.

In a more preferred embodiment of the present invention, the spasticmuscle is a smooth or striated muscle. Target tissue for the neurotoxiccomponent in the treatment of muscle hyperactivity disorders can be inprinciple all striated and smooth muscles of the human body as describedin Sobotta, Johannes: Atlas der Anatomie des Menschen. 22. Auflage. Band1 und 2. Urban & Fischer, 2005. The muscles mentioned in this referenceare incorporated herein by reference. The methods of the presentinvention may target any of these muscles.

The present invention also relates to a method of reducing facial linesor wrinkles of the skin or of removing facial asymmetries, the methodcomprising administering to an individual a composition comprising aneffective amount of a neurotoxic component of a Clostridium botulinumtoxin complex, the composition being devoid of any other proteincomponent of the Clostridium botulinum toxin complex, wherein (a) theindividual is a human; (b) the composition is administered bysubcutaneous or intramuscular injection into, or in vicinity of, one ormore facial muscles or muscles involved in the formation of the wrinkleof the skin or the asymmetry; and (c) the composition is administered atan interval of less than three months, the interval comprising a firsttreatment and a second treatment, wherein the amount administered in thesecond treatment can be lower, higher or identical to the amountadministered in the first treatment.

This method of the present invention allows treating facial muscles orwrinkles of a patient's skin or a facial asymmetry. Typically, smalleramounts of neurotoxic component are used in such cosmetic treatment.Such amounts are preferably in the range of 1 to 5, 5 to 10, 10 to 20 or20 to 50 Units. Such total amounts may be administered on the same dayor on a subsequent day of treatment. For example, during a firsttreatment session a first fraction of the dose may be administered. Thisfirst fraction is preferably a suboptimal fraction, i.e. a fraction,which does not remove the wrinkles or skin lines completely. During oneor more treatment sessions, the remaining fraction of the total dose maybe administered.

In a preferred embodiment of the present invention, the composition isinjected into the frown lines, horizontal forehead lines, crow's feet,perioral folds, mental ceases, popply chin, and/or platysmal bands.

In another preferred embodiment of the present invention, said muscle isselected from the group consisting of the following muscles: spleniuscapitis, sternocleidomastoid, scalene complex, levator scapulae,semispinalis, longissimus capitis, longissimus cervicis, multifidus,obliqus capitis inferior, obliqus capitis superior, rectus capitisposterior major, rectus capitis posterior minor, trapezius/parshorizontalis, trapezius/pars cervicalis, suprahyoidal muscles,infrahyoidal muscles, digastricus, pterygoideus medialis, pterygoideuslateralis, masseter, temporalis, orbicularis oculi, nasalis, procerus,corrugator supercilii, depressor anguli oris, depressor labiiinferioris, frontalis, levator labii superioris, levator labiisuperioris alaeque nasi, orbicularis oris, risorius, zygomaticusminor,zygomaticus major, deltoideus, triceps brachii, brachioradialis, bicepsbrachii, pronator quadratus pronator teres, flexor carpi radialis,flexor carpi ulnaris, flexor pollicis longus, opponens interossei,lumbricales, adductor pollicis, flexor pollicis brevis, flexor digitorumsuperficialis flexor digitorum profundus, adductor group, quadricepsfemoris, hamstrings, triceps surae, tibialis posterior, flexor hallucislongus, tibialis anterior, extensor hallucis longus, extensor digitorumlongus, flexor hallucis brevis, flexor digitorum brevis, paraverterbalmuscles.

The neurotoxic component referred to herein above, may be part of acomposition or pharmaceutical composition. This pharmaceuticalcomposition may contain additional pharmaceutically active components.“Pharmaceutical composition” is a formulation in which an activeingredient for use as a medicament or diagnostic is contained orcomprised. Such pharmaceutical composition may be suitable fordiagnostic or therapeutic administration (i.e. by intramuscular orsubcutaneous injection) to a human patient. The pharmaceuticalcomposition may be lyophilized or vacuum dried, reconstituted, or insolution When reconstituted it is preferred that the reconstitutedsolution is prepared adding sterile physiological saline (0.9% NaCl).

Such composition may comprise additional components such as a pH buffer,excipient, diluent, cryoprotectant and/or stabilizer.

“pH buffer” refers to a chemical substance being capable to adjust thepH value of a composition, solution and the like to a certain value orto a certain pH range.

“Stabilizing”, stabilizes” or “stabilization” means that the activeingredient, i.e., the neurotoxic component in a reconstituted or aqueoussolution pharmaceutical composition has greater than about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, and up to about 100% of the toxicity thatthe biologically active neurotoxic component had prior to beingincorporated into the pharmaceutical composition. The activity of thepreparation may be determined as described elsewhere herein.

“Cryoprotectant” refers to excipients which result in the activeingredient, i.e., a neurotoxic component in a reconstituted or aqueoussolution pharmaceutical composition has greater than about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, and up to about 100% of the toxicity thatthe biologically active neurotoxic component had prior to beingfreeze-dried in the pharmaceutical composition. The activity of thepreparation may be determined as described elsewhere herein.

Examples of such stabilizers are gelatin or albumin, preferably of humanorigin or obtained from a recombinant source. The stabilizers may bemodified by chemical means or by recombinant genetics. In a preferredembodiment of the present invention, it is envisaged to use alcohols,e.g., inositol, mannitol, as cryoprotectant excipients to stabilizeproteins during lyophilization.

In a more preferred embodiment of the present invention, the stabilizermay be a non-proteinaceous stabilizing agent comprising hyaluronic acidor polyvinylpyrrolidone or polyethyleneglycol or a mixture of two ormore thereof. Such composition is considered to be a safer compositionpossessing remarkable stability.

In a more preferred embodiment of the present invention, thepharmaceutical composition may comprise the neurotoxic component and ahyaluronic acid or a polyvinylpyrrolidone or a polyethleneglycol, suchcomposition being optionally pH stabilized by a suitable pH buffer, inparticular by a sodium acetate buffer, and/or a cryoprotectantpolyalcohol.

Whether or not the pharmaceutical composition comprises, beside theneurotoxin component additional components such as albumin, hyaluronicacid, a polyvinylpyrrolidone and/or a polyethyleneglycol stabilizer, thepharmaceutical composition retains its potency substantially unchangedfor six month, one year, two year, three year and/or four year periodswhen stored at a temperature between about +8° C. and about −20° C.Additionally, the indicated pharmaceutical compositions may have apotency or percent recovery of between about 20% and about 100% uponreconstitution.

A pharmaceutical composition within the scope of the present inventionmay include the neurotoxic component one or more additional components.Preferably, the pharmaceutical compositions disclosed herein, has a pHof between about 4 and 7.5 when reconstituted or upon injection, morepreferably between about pH 6.8 and pH 7.6 and most preferably betweenpH 7.4 and pH 7.6. Generally, the pharmaceutical composition of thepresent invention comprises neurotoxic component in a quantity of about6 pg to 30 ng, Preferably, the neurotoxic component has a biologicalactivity of 50 to 250 LD₅₀ units per ng neurotoxic component, asdetermined in a mouse LD₅₀ assay. More preferably, the neurotoxiccomponent has a biological activity of about 150 LD₅₀.

The pharmaceutical composition of the present invention may comprise aneurotoxin, and a hyaluronic acid. The hyaluronic acid stabilizes theneurotoxin. The pharmaceutical compositions disclosed herein may have apH of between about 4 and 7.5 when reconstituted or upon injection. Thehyaluronic acid in the instant pharmaceutical composition is preferablycombined with the instant neurotoxic component in a quantity of 0.1 to10 mg, especially 1 mg hyaluronic acid per ml in a 200 U/ml botulinumtoxin solution. More preferably, the subject solution also contains a1-100 mM, especially 10 mM sodium acetate buffer.

In another preferred embodiment, the composition may contain apolyalcohol as cryoprotectant Examples of polyalcohols that might beused include, e.g., inositol, mannitol and other non-reducing alcohols.

In particular those embodiments of the present invention'spharmaceutical composition not comprising a proteinaceous stabilizer,preferably do not contain trehalose or maltotriose or related sugar orpolyhydroxy compounds which are sometimes used as cryoprotectants.

The polyvinylpyrrolidone in the instant pharmaceutical composition ispreferably combined with the instant neurotoxic component in a quantityof 10 to 500 mg, especially 100 mg polyvinylpyrrolidone per ml in a 200U/ml botulinum toxin solution. More preferably, the subject solutionalso contains a 1-100 mM, especially 10 mM sodium acetate buffer.

The polyethyleneglycol in the instant pharmaceutical composition ispreferably combined with the instant neurotoxic component in a quantityof 10 to 500 mg, especially 100 mg polyethyleneglycol per ml in a 200U/ml botulinum toxin solution. More preferably, the subject solutionalso contains a 1-100 mM, especially 10 mM sodium acetate buffer.

Thus, the instant invention encompasses in a more preferred embodiment aneurotoxic component formulated in a pharmaceutical composition, whichcontains a hyaluronic acid stabilizer or a polyvinylpyrrolidonestabilizer or a polyethyleneglycol stabilizer. Additionally, thepharmaceutical composition may contain a sodium acetate buffer systemand/or an alcoholic cryoprotectant.

The following examples are provided by means of illustration only, andare not intended to be limiting.

EXAMPLE 1 Botulinum Toxin Therapy for Treatment of Cervical Dystonia

A 45 year-old male patient suffering from cervical dystonia is evaluatedfor botulinum toxin therapy. After all appropriate examinations aninjection scheme is constructed and botulinum toxin free of complexingproteins is applied accordingly in a total dose of 300 MU. Onre-evaluation after 2 weeks the symptomatology is improved, but there isa need to include additional target muscles and to increase thebotulinum toxin dose in initially injected target muscles. Two weekslater the patient is re-evaluated again and the treatment result isoptimal.

Adverse effects do not occur. So far, on 7 subsequent injection seriesthe treatment results are maintained without any indication ofantibody-induced therapy failure.

EXAMPLE 2 Botulinum Toxin Therapy for Treatment of Blepharospasm. ShortDuration of Action

A 61 year-old female patient suffering from blepharospasm is treatedwith a medicament containing the neurotoxic component of the presentinvention, free of complexing proteins in a total dose of 48 MU withexcellent results. 4 weeks after the injections with the neurotoxiccomponent the effect begins to wane. After 2 more weeks the effect ofthe treatment has almost completely ceased. Re-Injections are performed7 weeks after the initial injection series. Therapy with the neurotoxiccomponent is repeated in the initial dose and with identical effects.Therapy with the neurotoxic component is continued for 6 subsequentinjection series with excellent therapeutic results and without anyindication of antibody-induced therapy failure.

EXAMPLE 3 Botulinum Toxin Therapy for Treatment of GeneralisedSpasticity

High Dose Application.

A 35 year-old male patient suffering from hypoxic brain damage withgeneralized spasticity The neurotoxic component of the presentinvention, free of complexing proteins in a total dose of 750 MU, isadministered in three aliquots of 250 MU given with 1 day intervals 2weeks after the application the condition has improved substantially.Adverse effects neither local nor regional nor systemic, cannot bedetected. On 7 subsequent injection series the therapeutic effect isstable without occurrence of adverse effects. There is no indication ofantibody-induced therapy failure.

EXAMPLE 4 Cosmetic Use of Botulinum Toxin. Difficulties in Constructingthe Injection Scheme and Short Duration of Action

A 40 year old female client presenting with muscular frowning lines andhorizontal frontal lines was treated with 20 MU of botulinum toxin freeof complexing proteins (i.e. the neurotoxic component of the presentinvention). 2 weeks later there is an improvement of the symptomatology,but additional injection of 20 MU of botulinum toxin are necessary. 2weeks later the outcome is fully satisfactory for the patient. 4 weekslater the favorable effect starts to wane, so that botulinum toxinre-injections in a total dose of 40 MU become necessary. So far, theclient has undergone 4 subsequent injection series with total doses of40 MU each. There is no indication of antibody-induced therapy failure.

1. A method of treating a disease or condition caused by or associatedwith hyperactive cholinergic innervation of muscles or exocrine glandsin a patient, the method comprising administering a compositioncomprising an effective amount of a neurotoxic component of aClostridium botulinum toxin complex, the composition being devoid of anyother protein component of the Clostridium botulinum toxin complex,wherein (a) the patient is a human, (b) the composition is administeredby injection, and (c) the composition is administered at an interval ofless than three months, the interval comprising a first treatmentsession and a second treatment session, wherein the amount administeredin the second treatment session can be lower, higher or identical to theamount administered in the first treatment.
 2. The method of claim 1,wherein the second treatment is performed in order to improve thetreatment effect of the first treatment.
 3. The method of claim 1,wherein (a) the patient is a patient with a severe movement disorder orsevere spasticity and (b) the effective amount administered exceeds 500U of neurotoxic component in adults or exceeds 15 U/kg body weight inchildren.
 4. The method of claim 3, wherein the amount which exceeds 500U is a total amount in adults or exceeds 15 U/kg body weight in childrenand wherein the amount is administered by (a) injecting a first fractionof this amount during a first treatment session; and (b) injecting theremaining fraction during one or more subsequent treatment session(s),wherein the subsequent treatment session(s) is/are scheduled at leastone day after the first treatment session.
 5. The method of claim 1,wherein the patient is a human, who has been treated with a Clostridiumbotulinum toxin but who complains about a decrease of the treatmenteffect and who requires an additional treatment within 3 months of aprevious treatment.
 6. The method of claim 1, wherein the hyperactivegland is an autonomic exocrine gland and wherein the composition isinjected into or in the vicinity of such gland.
 7. The method of claim6, wherein the (a) gland is selected from the group consisting of sweatglands, tear glands, salivary glands and mucosal glands; or (b) gland isassociated with a disease or condition selected from the groupconsisting of Frey syndrome, Crocodile Tears syndrome, axillarhyperhidrosis palmar hyperhidrosis, plantar hyperhidrosis, hyperhidrosisof the head and neck, hyperhidrosis of the body, rhinorrhea, or relativehypersalivation in patients with stroke, Parkinson's disease orAmyotrophic Lateral Sclerosis.
 8. A method of treating a disease orcondition caused by hyperactive cholinergic innervation of a muscle in apatient, the method comprising administering a composition comprising aneffective amount of a neurotoxic component of a Clostridium botulinumtoxin complex, the composition being devoid of any other proteincomponent of the Clostridium botulinum toxin complex, wherein (a) thepatient is a human with a severe movement disorder or severe spasticity;(b) the composition is administered by injection; and (c) the effectiveamount administered exceeds 500 U of neurotoxic component in adults orexceeds 15 U/kg body weight in children.
 9. The method of claim 8,wherein the amount which exceeds 500 U is a total amount in adults orexceeds 15 U/kg body weight in children and wherein the amount isadministered by (a) injecting a first fraction of this amount during afirst treatment session; and (b) injecting the remaining fraction duringone or more subsequent treatment session(s), wherein the subsequenttreatment session is scheduled at least one day after the firsttreatment session.
 10. The method of claim 8, wherein the composition isadministered at an interval of less than three months, the intervalcomprising a first treatment session and a second treatment session,wherein the amount administered in the second treatment session can belower, higher or identical to the amount administered in the firsttreatment session.
 11. The method of claim 1, wherein the disease orcondition is or involves dystonia of a muscle.
 12. The method of claim11, wherein the dystonia (a) is selected from the group consisting of(1) cranial dystonia including blepharospasm oromandibular dystonia ofthe jaw opening or jaw closing type, bruxism, Meige syndrome, lingualdystonia, apraxia of eyelid opening, (2) cervical dystonia includingantecollis, retrocollis, laterocollis, torticollis (3) pharyngealdystonia, (4) laryngeal dystonia including spasmodic dysphonia of theadductor type or of the abductor type, spasmodic dyspnea (5) limbdystonia including arm dystonia such as task specific dystonias,including writer's cramp, musician's cramps or golfer's cramp, legdystonia involving thigh adduction, thigh abduction, knee flexion, kneeextension ankle flexion, ankle extension or equinovarus deformity, footdystonia involving striatal toe, toe flexion or toe extension, axialdystonia such as Pisa syndrome or belly dancer dystonia, segmentaldystonia, hemidystonia or generalized dystonia, (6) dystonia in Lubag,(7) dystonia in corticobasal degeneration (8) tardive dystonia, (9)dystonia in spinocerebellar ataxia, (10) dystonia in Parkinson'sdisease, (11) dystonia in Huntington's disease, (12) dystonia inHallervorden Spatz disease, (13) dopa-induced dyskinesias/dopa-induceddystonia, (14) tardive dyskinesias/tardive dystonia, (15) paroxysmaldyskinesias/dystonias (kinesiogenic, non-kinesiogenic, action-induced);or (b) involves a clinical pattern selected from the group consisting oftorticollis, laterocollis, retrocollis, anterocollis, flexed elbow,pronated forearm, flexed wrist, thumb-in-palm or clenched fist.
 13. Themethod of claim 11, wherein the muscle is selected from the groupconsisting of Ipsilateral splenius, contralateral sternocleidomastoid,ipsilateral sternocleidomastoid splenius capitis, scalene complex,levator scapulae, postvertebralis, ipsilateral trapezius, levatorscapulae, bilateral splenius capitis, upper trapezius, deeppostvertebralis, bilateral sternocleidomastoid, scalene complex,submental complex brachioradialis, biceps brachialis, pronator quadratuspronator teres, flexor carpi radialis, flexor carpi ulnaris, flexorpollicis longus, adductor pollicis, flexor pollicis brevis/opponens,flexor digitorum superficialis, flexor digitorum profundus.
 14. Themethod of claim 1, wherein the disease or condition is or involvesspasticity of a muscle.
 15. The method of claim 14, wherein thespasticity is or is associated with (1) a spastic condition inencephalitis and myelitis relating to (a) autoimmune processes includingrespect to multiple sclerosis, transverse myelitis, Devic syndrome, (b)viral infections, (c) bacterial infections, (d) parasitic infections or(e) fungal infections, (2) hereditary spastic paraparesis, (3)postapoplectic syndrome resulting from hemispheric infarction, brainsteminfarction or, myelon infarction, (4) a central nervous system traumainvolving e.g. a hemispheric lesion, a brainstem lesion, a myelonlesion, (5) a central nervous system hemorrhage such as an intracerebralhemorrhage, a subarachnoidal hemorrhage, a subdural hemorrhage or anintraspinal hemorrhage, or (6) a neoplasia, e.g. a hemispheric tumor, abrainstem tumors or a myelon tumor or (7) post-stroke spasticity, or (9)spasticity caused by cerebral palsy.
 16. The method of claim 14, whereinthe muscle is a smooth or striated muscle.
 17. A method of reducingfacial lines or wrinkles of the skin or of removing facial asymmetries,the method comprising administering to an individual a compositioncomprising an effective amount of a neurotoxic component of aClostridium botulinum toxin complex, the composition being devoid of anyother protein component of the Clostridium botulinum toxin complex,wherein (a) the individual is a human; (b) the composition isadministered by subcutaneous or intramuscular injection into, or invicinity of, one or more facial muscles or muscles involved in theformation of the wrinkle of the skin or the asymmetry; and (c) thecomposition is administered at an interval of less than three months,the interval comprising a first treatment session and a second treatmentsession, wherein the amount administered in the second treatment sessioncan be lower, higher or identical to the amount administered in thefirst treatment session.
 18. The method of claim 17, wherein thecomposition is injected into the frown line, horizontal forehead line,crow's feet, nose perioral fold, mental ceases, popply chin, and/orplatysmal bands.
 19. The method of claim 17, wherein the injected muscleis selected from the group consisting of corrugator supercillii,orbicularis oculi, procerus, venter frontalis of occipitofrontalis,orbital part of orbicularis oculi, nasalis, upper lip: orbicularis oris,lower lip: depressor angulis oris, mentalis and platysma, which musclesare involved in forming such lines.
 20. The method of claim 1, whereinthe neurotoxic component is selected from the group consisting of typeA, B, C, D, E, F, G or a mixture thereof.